This invention relates to genetic screening methods useful in isolating genes and identifying unknown functions of genes or unknown functional links between genes as well as in identifying molecules having gene-specific lethal properties.
The following publications may be relevant to the invention, and are referred to in the specification by number:
1. Bender and J. R. Pringle, Mol. Cell. Biol. 11, 1295 (1991);
2. V. Doye and E. C. Hurt, Trends Genetics 11, 235 (1995);
3. Koshland, D. et al, Cell 40,393 (1985).
With continued progress in the Human Genome Project as well as the initiation of the Mouse/Rat Genome Projects, and the sequencing of the majority of the human cDNAs, the elucidation of gene function has become a major priority. High throughput screening methods are required in order to determine the functions of large numbers of genes in an efficient manner.
Screening methods are also required for discovering novel gene-specific drugs. In the search for such drugs, it would be advantageous to be able to elucidate the interaction between specific chemical reagents and one or more genes in a high throughput format. This is particularly relevant with respect to cancer drugs.
A synthetic or synergistic lethality screening method has previously been described in yeast cells (1,2). The essence of this screen in yeast, is the ability to identify nonallelic and nonessential mutation/s that are lethal in combination with a nonessential mutation in a gene of interest (i.e. synthetic lethality). A wild-type copy of the gene of interest, on an episomal plasmid, is introduced into cells which are null for expression of this gene. Random chemical mutagenesis of the entire yeast genome within these cells may inactivate a gene which is synthetically lethal with the gene of interest. Under these conditions, retention of the episomal plasmid, which is otherwise spontaneously lost, and expression of the gene of interest become essential for survival (i.e. survival plasmid). Plasmid loss or retention is detected by changes in colony pigmentation, due to the presence on the plasmid of a wild-type gene whose product is essential for red pigment accumulation (3). This genetic method is very powerful as it can reveal not only interactions between gene products with direct physical contacts, but also interactions along the same or parallel pathways.
A synthetic lethality screen in non-yeast eukaryotic cells in general and in human cells in particular, has not yet been described.
In an effort to expand the arsenal of genetic tools which can be applied to other eukaryotic biological systems, it has now been discovered that the concept of the yeast synthetic lethality screen can be applied to non-yeast eukaryotic cells, particularly to mammalian cells and most particularly to human cells.
An object of the present invention is to provide a screening method useful in identifying molecules having gene-specific cell-lethal properties.
A further object of the present invention is to provide a screening method useful in isolating genes and identifying unknown functions of genes or unknown functional links between genes.
In a first aspect of the invention, there is provided a method for screening a chemical library comprising a plurality of molecule types in non-yeast eukaryotic cells having a genome, in order to identify a molecule type having a gene-specific lethal property in the cell, the genome comprising a gene of interest which carries a non-lethal mutation therein.
The method comprises the following steps:
(a) transfecting a first reporter gene into the cells, and selecting clones stably expressing the reporter gene;
(b) introducing into the cells an episome comprising a functioning copy of the gene of interest, a second reporter gene, a dominant selectable marker, an origin of DNA replication and a nuclear antigen gene essential for replication of the episome within the cells, wherein the episome is spontaneously lost from the cells, and growing the cells in the presence of a selection compound which selects for the dominant selectable marker;
(c) selecting cell clones stably expressing the second reporter gene and the functioning copy of the gene of interest;
(d) seeding of the cells into a plurality of cell chambers and removing the selection compound;
(e) adding a molecule type of the chemical library to each of the plurality of cell chambers, measuring expression of the first and second reporter genes in the chambers, and calculating the ratio of the measured expressions, thereby indicating retention of the episome; and
(f) identifying a cell chamber in which the episome is retained, thus identifying a molecule type having a gene-specific lethal property in the cell.
In this aspect of the invention, chemical reagent induced synthetic lethality (i.e. chemical synthetic lethality) identifies biochemical inhibitors or drugs whose lethal effect is dependent on the full or partial inactivation of a specific gene (i.e. gene of interest) within its cellular milieu.
In an alternate embodiment of this aspect of the invention, there is provided a method for screening a chemical library comprising a plurality of molecule types in non-yeast eukaryotic cells having a genome, in order to identify a molecule type having a gene-specific lethal property in the cell, the genome comprising a wild-type gene of interest.
The method comprises the following steps:
(a) transfecting a first reporter gene into the cells, and selecting clones stably expressing the reporter gene;
(b) introducing into the cells an episome comprising a dominant-negative mutant of the gene of interest, a second reporter gene, a dominant selectable marker, an origin of DNA replication and a nuclear antigen gene essential for replication of the episome within the cells, wherein the episome is spontaneously lost from the cells, and growing the cells in the presence of a selection compound which selects for the dominant selectable marker;
(c) selecting cell clones stably expressing the second reporter gene and the dominant-negative mutant of the gene of interest;
(d) seeding of the cells into a plurality of cell chambers and removing the selection compound;
(e) adding a molecule type of the chemical library to each of the plurality of cell chambers, measuring expression of the first and second reporter genes in the chambers, and calculating the ratio of the measured expressions, thereby indicating retention of the episome; and
(f) identifying a cell chamber in which the episome is retained, thus identifying a molecule type having a gene-specific lethal property in the cell.
In a second aspect of the invention, there is provided a method for screening a collection of DNA molecules selected from the group consisting of antisense cDNA, truncated cDNA, full-length cDNA and genomic DNA, in order to identify from among them one or more modulators of gene function which are synergistically lethal to a non-yeast eukaryotic cell together with an incapacitated gene of interest, the cell having a genome which comprises the incapacitated gene of interest.
The method of this aspect of the invention comprises the following steps:
(a) transfecting a first reporter gene into the cell, and selecting clones stably expressing the reporter gene;
(b) introducing into the cells an episome comprising a functioning copy of the gene of interest, a second reporter gene, a first dominant selectable marker, an origin of DNA replication and a nuclear antigen gene essential for replication of the episome within the cells, wherein the episome is spontaneously lost from the cells, and growing the cells in the presence of a selection compound which selects for the first dominant selectable marker;
(c) selecting cell clones stably expressing the second reporter gene and the functioning copy of the gene of interest;
(d) incorporating each of the DNA molecules into vector vehicles containing a second dominant selectable marker gene;
(e) transfecting the cells with the vector vehicles so that each transfected cell contains on the average approximately one to several of the molecules, and allowing expression of the molecules in the cells;
(f) seeding the transfected cells into a plurality of cell chambers under conditions of one stably transfected cell or less per chamber, and initiating conditions for selection of the second dominant marker, while removing the selection compound for the first dominant selectable marker;
(g) measuring expression of the first and second reporter genes in the chambers, and calculating the ratio of the measured expressions, thereby indicating retention of the episome; and
(h) identifying a cell chamber in which the episome is retained, thus identifying a DNA molecule which is a modulator of gene function.
In a further embodiment of this aspect of the invention, there is provided a method for screening a collection of DNA molecules selected from the group consisting of antisense cDNA, truncated cDNA, full-length cDNA and genomic DNA, in order to identify among them one or more modulators of gene function which are synergistically lethal to a non-yeast eukaryotic cell, the cell having a genome which comprises a wild-type gene of interest.
The method of this embodiment comprises the following steps:
(a) transfecting a first reporter gene into the cell, and selecting clones stably expressing the reporter gene;
(b) introducing into the cells an episome comprising a dominant-negative mutant of the gene of interest, a second reporter gene, a first dominant selectable marker, an origin of DNA replication and a nuclear antigen gene essential for replication of the episome within the cells, wherein the episome is spontaneously lost from the cells, and growing the cells in the presence of a selection compound which selects for the first dominant selectable marker;
(c) selecting cell clones stably expressing the second reporter gene and the dominant-negative mutant of the gene of interest;
(d) incorporating each of the DNA molecules into vector vehicles containing a second dominant selectable marker gene;
(e) transfecting the cells with the vector vehicles so that each transfected cell contains on the average approximately one to several of the molecules, and allowing expression of the molecules in the cells;
(f) seeding the transfected cells into a plurality of cell chambers under conditions of one stably transfected cell or less per chamber, and initiating conditions for selection of the second dominant marker, while removing the selection compound for the first dominant selectable marker;
(g) measuring expression of the first and second reporter genes in the chambers, and calculating the ratio of the measured expressions, thereby indicating retention of the episome; and
(h) identifying a cell chamber in which the episome is retained, thus identifying a DNA molecule which is a modulator of gene function.
In this aspect of the invention, synthetic lethality imposed by either a GSE (defined below) or by an overexpressed full-length cDNA (i.e. genetic synthetic lethality) identifies gene function or functional links between genes.
The following terms used in the present specification have the indicated definitions:
non-essential genexe2x80x94a gene whose function is non-essential to the viability of the cell, either because it is dispensible for cell metabolism or due to the existence of one or more other genes which functionally overlap with it.
non-lethal mutationxe2x80x94a mutation within a non-essential gene or a defect within an essential gene which is partial and thus leaves the cell viable.
gene of interestxe2x80x94a specific gene which is either non-essential for viability or an essential gene carrying a non-lethal mutation. Its function may be known or unknown.
synthetic or synergistic lethalityxe2x80x94a lethal cell phenotype which is the result of either the synergistic incapacitation of two (or more) genes, or due to the overexpression of one gene on the background of the incapacitation of the gene of interest. Either one of these two conditions may also require the overexpression and/or underexpression of other gene(s). The incapacitation of activity may be full or only partial. The incapacitation may be as a result of a resident mutation, or due to an externally inserted element, such as a truncated cDNA, an antisense cDNA molecule or a chemical reagent.
Episomal survival plasmidxe2x80x94a gene vehicle/vector which carries either a functioning copy or a dominant-negative mutant of a gene of interest. The plasmid is not incorporated into the genome of the cell, and yet can autonomously replicate within the cell (i.e. an episome). The plasmid is spontaneously gradually lost from the cell population.
genetic suppressor element (GSE)xe2x80x94a nucleic acid capable of suppressing genetic expression in a dominant-negative fashion. Examples of GSEs are antisense cDNA, truncated sense cDNA, and other forms of mutated DNA.
modulators of gene expressionxe2x80x94a group of DNA molecules which affect gene expression of the host cells. These DNA molecules are either GSEs or overexpressed wild-type genes.
chemical libraryxe2x80x94a group of different chemical reagents which may comprise synthetic as well as natural compounds. Each of the different types of reagents may be referred to at times as xe2x80x98molecule typesxe2x80x99. This definition also includes, but is not limited to, synthetic antisense DNA oligonucleotides which may also be modified (phosphorothioate antisense oligodeoxynucleotides, chimeric oligodeoxynucleotides, etc.).
The method of the invention is based on the screening of a cell population for either loss of gene function or gene overexpression resulting in lethality. The survival plasmid carries the genetic information which is necessary for cell survival under the specific conditions of the imposed synthetic lethality. This results in a strong selective advantage for retaining the episomal survival plasmid which would otherwise be lost from the cell. In this manner, the phenotype of lethality may be identified by the maintenance of the plasmid in an otherwise non-viable cell.
Synthetic lethality is dependent on two independent events of gene incapacitation, or a combination of the incapacitation of the gene of interest, and overexpression of the other gene in its non-mutated form. The gene incapacitation may be either by genetic means or by an external specific chemical inhibitor. Thus, if the nature of one of the events is known, the nature of the other event can be surmised as being overlapping, complementing or antagonizing the first event. In this way, an unknown function may be identified.
The method of the invention differs from the synthetic lethality screen previously described in yeast in the following respects:
(1) Synthetic lethality as disclosed in yeast is recognized by the visible color of yeast colonies grown on agar within petri dishes. The majority of colonies exhibit the appearance of white sectors within red colonies, while a synthetic lethal condition prevents the appearance of white sectors in a primarily red colony. The accumulation of red pigment is enabled by the reporter gene acting together with other genes.
In contrast, the method of the invention involves the seeding of human/mammalian cells into microtiter plates, and the periodic measurement in a fluorescent plate reader of the double-label fluorescent ratio of two fluorescent proteins. Retention over time of a high ratio in the readings of a fluorescent variant encoded by the survival plasmid to a second fluorescent variant produced from a chromosomally integrated gene, indicates selection for maintenance of the survival plasmid and thus a synthetic lethality condition. The fluorescence is a direct product of the reporter gene.
(2) Synthetic lethality is imposed in yeast by randomly mutagenizing the whole yeast genome with a chemical mutagen, thus leading to random gene inactivation. In contrast, in the present invention, synthetic lethality is achieved by either a chemical inhibitor (chemical synthetic lethality) or a genetic incapacitation (genetic synthetic lethality). The latter involves overexpressing sense cDNA libraries or GSE libraries, either one of which is incorporated into episomal plasmids (J. L. Yates, N. Warren and B. Sugden, Nature 313, 812 (1985); L. P. Deiss and A. Kimchi, Science 252, 117 (1991)), retroviral vectors (A. V. Gudkov et al., Proc. Natl. Acad. Sci. USA 90, 3231 (1993)), chimeric transposable elements (Z. Ivics et al, Cell 91,501 (1997)) or episomal viral vectors.
(3) Identification of the gene which is synthetic lethal with the gene of interest is performed in yeast by first isolating those colonies in which the red pigment was maintained and no white sectors appear. Those colonies putatively harbor a chromosomally mutated gene which is synthetic lethal with the gene of interest. Those yeast colonies are transfected by a normal yeast genomic library incorporated into a yeast multi-copy plasmid. Those transformants transfected by and expressing a wild-type copy of the chromosomally mutated gene, no longer sustain a synthetic lethality condition, and therefore no longer need to retain the survival plasmid. Those few colonies are recognized by the appearance of white sectors, from which the plasmid DNA is extracted, transformed into bacteria and further analyzed for the identity of the yeast gene insert by standard recombinant DNA methods.
Identification of the genetic element which confers the synthetic lethal phenotype in human/mammalian cells of the present invention, on the other hand, does not require a further transfection with a normal gene/cDNA library. This is because, unlike in the yeast method, gene incapacitation is not achieved by mutagenizing the endogenous resident cell genome but rather by an exogenous DNA element working either in a dominant-negative fashion or by overexpression of a wild-type cDNA. Accordingly, the external genetic element conferring the synthetic lethality is recovered by either one of two approaches, depending on the type of vector/vehicle used: (a) an episomal plasmid, or a chimeric episomal virus are rescued by Hirt supernatant extract mediated bacterial transformation or virus particle purification, respectively; (b) chromosomally integrated GSE or a wild-type sense cDNA library incorporated into either a retroviral vector or a chimeric transposable element, are recovered by PCR on genomic DNA.
The availability of a large number of mutant human cell lines derived from genetic disorders on the one hand, and the ability to employ homologous recombination for gene disruption in somatic human cells on the other, constitutes a large reservoir of recipient cells and genes of interest.
The cells which may be used in the method of the invention are non-yeast eukaryotic cells. Preferably they are human cells, but the same principle may be applied to e.g., rodent cells harboring a survival plasmid with the appropriate replication properties.
The survival plasmid contains a reporter gene so as to enable determination of the presence of the plasmid in the cells. The product of the reporter gene may be any detectable molecule, such as the following biosensors: luciferin (luciferase substrate); aequorin; Fluo-3/acetoxymethyl (esterase substrate); FDG (xcex2-gal substrate); or CCF2 which is a xcex2-lactamase substrate [J. E. Gonzxc3xa1lez and P. A. Negulescu, Curr. Opin. Biotechnol. 9, 624 (1998)]. Preferably, the reporter gene encodes a fluorescent protein whose expression can be distinguished from that of a second fluorescent protein marking the cell number. Non overlapping excitation or emission spectra of the two fluorescent proteins allows for double-label fluorescence measurement.
Accordingly the cells are also made to incorporate in their genome a second reporter gene which indicates the number of cells. By comparing the signals obtained from the two reporter genes, a relative ratio between the number of survival plasmids and the number of cells may be determined.
The methods of the invention may be carried out using conventional systems for growing, scanning and sorting cells, such as microtiter plates, 96-well, 384-well or other high-density microplates, a microplate fluorescent reader, and a fluorescent activated cell sorter (FACS). The methods are especially useful in high throughput screening, where automation allows for the rapid screening of large number of chemicals as well as the full spectrum of mammalian genes and their respective GSEs.
Examples of GSEs are dominant-negative genetic elements such as truncated sense cDNA and antisense cDNA.
The present invention may be used in a number of applications.
The first aspect of the invention should prove advantageous in the search of drugs which synergize with particular gene deficiencies to cause cell lethality. A special application of this aspect would be to look for chemicals which kill either a benign or cancerous cell growth in a defined genetic milieu where the chemical is synthetic lethal with a particular mutant gene.
The second aspect of the invention is useful in identifying human genes whose under or over-expression causes lethality of human cell lines with defined genetic abnormalities. Such genes are obviously potential targets for drugs aimed at eliminating the affected cells/tissue. The application of this approach to human tumor-derived cell lines, is particularly amenable to identification of targets for cancer therapy.
Above and beyond the identification of gene targets of therapeutic interest in defined genetic background, the invention should prove useful as a tool for basic research. In particular, the invention may enable researchers to rapidly screen large sets of gene products for functional interactions and helps define genetic pathways within the cell (2).
The method using rodent cells should be useful as a model for human genetic traits and responses in drug development and disease research. For example, mutant mice generated by either homologous recombination or tagged random mutagenesis, supply a large source of recipient mutated mouse embryo fibroblasts which, together with the methods of the invention, will greatly facilitate research and development of new drugs and therapeutic strategies for human beings.
Also included in the invention are kits for synthetic lethality screening. One such kit in accordance with the first aspect of the invention would preferably include an episomal survival plasmid and an integrating vector, each carrying a reporter gene, for a chemical synthetic lethality screen. A kit in accordance with the second aspect of the invention would preferably include the above genetic elements together with a library of GSEs or sense cDNAs incorporated within an episomal vector.